Compounds and methods to treat organophosphorus poisoning

ABSTRACT

Organophosphate (OP) nerve agents and pesticides are potent inhibitors of acetylcholinesterase (AChE). Though oxime nucleophiles can reactivate an AChE-phosphyl adduct, the adduct can undergo a reaction called aging, leading to an aged-AChE adduct. The invention provides compounds and methods that can be used to reactivate an aged-AChE adduct. Such compounds and methods are useful to counteract organophosphorus poisoning.

RELATED APPLICATION

This patent application claims the benefit of priority of U.S.Application Ser. No. 61/756,311 filed Jan. 24, 2013. The content of thisprovisional application is hereby incorporated herein in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 5 R21NS076430 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Organophosphorus (OP) agents have been used as both pesticides andchemical warfare agents for most of the past century (Mercey, G., etal., Acc. Chem. Res. 2012, 45, 756; and Jokanovic, M.; Prostran, M.Curr. Med. Chem. 2009, 16, 2177). Pesticides based on phosphate orthiophosphate agents (e.g. parathion, diazinon, or malathion) are orhave been used in domestic and commercial agriculture to protect cropsfrom a variety of destructive species. Despite numerous efforts to banor limit the use of OP pesticides, their continued use results in over200,000 fatalities and over 1 million instances of morbidity annuallydue to OP exposure. Phosphonate nerve agents, including sarin, soman,VX, and tabun, are extremely toxic and are considered a serious threatto national security due to their potential use in terrorist actions(See Millard, C. B.; et al., Biochem. 1999, 38, 7032; Sanson, B.; etal., J. Med. Chem. 2009, 52, 7593; Carletti, E.; et al., J. Med. Chem.2010, 53, 4002; Masson, P.; Nachon, F.; Lockridge, O. Chem. Bio.Interact. 2010, 187, 157; and Mercey, G.; et al., Acc. Chem. Res. 2012,45, 756).

These nerve agents elicit their acute toxicity by inhibiting theacetylcholinesterase (AChE), which results in an overstimulation ofmuscles due to the buildup of acetylcholine in the neuromuscularjunction. The ultimate result is muscle fasciculation and subsequentcardiopulmonary arrest (Mercey, G.; et al., Acc. Chem. Res. 2012, 45,756; and Jokanovic, M.; Prostran, M. Curr. Med. Chem. 2009, 16, 2177).Specifically, OPs inhibit AChE by covalently bonding to the serineresidue in the catalytic triad of the active site (FIG. 1). The welldocumented and tremendous affinity of OP agents for the active cite ofAChE is a result of OP's structural similarity to the tetrahedraltransition state of the cognate hydrolysis reaction of acetylcholine(Quinn, D. M. Chem. Rev. 1987, 87, 955).

There are several potential treatments for both acute and chronicorganophosphate poisoning. Most include a nucleophilic oxime toreactivate the enzyme along with atropine as an acetylcholine receptorantagonist and diazepam to treat seizures that arise from inhibition ofAChE in the CNS (Mercey, G.; et al., Acc. Chem. Res. 2012, 45, 756; andJokanovic, M.; Prostran, M. Curr. Med. Chem. 2009, 16, 2177). Ifadministered shortly after exposure, the oxime (e.g. 2-pyridine aldoximemethyl chloride, 2-PAM) is able to displace the bound OP and liberatethe serine residue (FIG. 1). (Kalisiak, J.; et al., J. Med. Chem. 2011,54, 3319; Kalisiak, J.; et al., J. Med. Chem. 2012, 55, 465; Mercey, G.;et al., Chem. Commun. 2011, 47, 5295; and Sit, R. et al., J. Bio. Chem.2011, 286, 19422.)

If administration of the oxime is delayed a process called aging occurswhere the enzyme bound phosphonate experiences a solvolytic loss of analkyl group (FIG. 1: R=isopropyl for sarin, pinacoyl for soman). SeeMillard, C. B.; et al., Biochem. 1999, 38, 7032; Sanson, B.; et al., J.Med. Chem. 2009, 52, 7593; Carletti, E.; et al., J. Med. Chem. 2010, 53,4002; Masson, P.; Nachon, F.; Lockridge, O. Chem. Bio. Interact. 2010,187, 157; and Mercey, G.; et al., Acc. Chem. Res. 2012, 45, 756. Theaged adduct is a phosphonate ester monoanion, which is intrinsicallyless reactive as an electrophile that the erstwhile neutral phosphonateester of the initial phosphonyl-AChE adduct. The aged adduct is furtherstabilized by several key interactions with the AChE active site, and isrefractory to oxime reactivation. See Millard, C. B.; et al., Biochem.1999, 38, 7032; Sanson, B.; et al., J. Med. Chem. 2009, 52, 7593;Carletti, E.; et al., J. Med. Chem. 2010, 53, 4002; and Masson, P.;Nachon, F.; Lockridge, O. Chem. Bio. Interact. 2010, 187, 157. Nocompounds have been reported that are able to recover AChE activity onceaging has occurred, and therefore there are no known antidotes againstaged AChE-OP adducts. See Mercey, G., et al., Acc. Chem. Res. 2012, 45,756; Kalisiak, J.; et al., J. Med. Chem. 2011, 54, 3319; Kalisiak, J.;et al., J. Med. Chem. 2012, 55, 465; Mercey, G.; et al., Chem. Commun.2011, 47, 5295; and Sit, R. et al., J. Bio. Chem. 2011, 286, 19422.

Currently there is a need for compounds and methods that can be used toreactivate the aged-AChE adduct. Such compounds and methods would beuseful to counteract organophosphorus poisoning.

SUMMARY OF THE INVENTION

Applicant has discovered a class of compounds and a method that can beused to reactivate an aged-AChE adduct. Because the aged complex haslong been thought of as a dead enzyme, this concept has been termed“resurrection” of the aged adduct (FIG. 1).

Accordingly, the invention provides a method for reactivating anaged-AChE adduct comprising contacting the aged-AChE adduct with analkylating agent that leads to reactivation of the aged-AChE adduct.

The invention provides a method for treating an animal (e.g. a mammal)suffering from organophosphorus poisoning comprising administering anamount of an alkylating agent effective to reactivate aged-AChE to theanimal. The method can further comprise administering an agent capableof reactivating AChE (e.g. a nucleophilic oxime) to the animal. Themethod can also further comprise administering an acetylcholine receptorantagonist (e.g. atropine) and or an antiseizure agent (e.g. diazepam)to the animal.

The invention provides a method for increasing acetylcholinesteraseactivity in an animal (e.g. an animal in need of such treatment)comprising administering an amount of an alkylating agent effective toreactivate aged-AChE to the animal. The method can further compriseadministering an agent capable of reactivating AChE (e.g. a nucleophilicoxime) to the animal. The method can also further comprise administeringan acetylcholine receptor antagonist (e.g. atropine) and or anantiseizure agent (e.g. diazepam) to the animal.

The invention also provides a compound of formula (I):

wherein:

R₁ is (C₁-C₃)alkyl, aryl(C₁-C₃)alkyl, or —Y—Z, wherein any (C₁-C₃)alkylis optionally substituted with one or more halo, and wherein any aryl isoptionally substituted with one or more groups independently selectedfrom halo, cyano, nitro, carboxy, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo;

each R₂ is independently H, halo, cyano, carboxy, —CH═N—OH,(C₁-C₃)alkyl, aryl, heteroaryl, aryl(C₁-C₃)alkyl, orheteroaryl(C₁-C₃)alkyl, wherein any (C₁-C₃)alkyl is optionallysubstituted with one or more halo, and wherein any aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom halo, cyano, nitro, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo;

each X is independently a suitable a counter ion;

Y is —(CH₂)_(n)— or —(CH₂)_(n)—O—(CH₂)_(n)—;

n is 1, 2, 3, 4, 5, or 6; and

Z is:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an organophosphorous agent (OP) inhibitingacetylcholinesterase (AChE) by covalently bonding to the serine residuein the catalytic triad of the active site to form an AChE-OP Adduct. Ifadministration of an oxime is delayed, a process called aging occurs,wherein the enzyme bound phosphonate experiences a solvolytic loss of analkyl group (Aging) to form an Aged Adduct. Reactivation (Resurection)of the Aged-Adduct to provide the AChE-OP Adduct (e.g. using a compoundor method of the invention) is also illustrated.

FIG. 2 illustrates the resurrection assay procedure (Example 3) forCompound 100 alkylation and reactivation of “aged” hAChE. The assay wascarried out at pH 7.3 and 27° C. Dilution concentrations of hAChE arerepresentative of a 100 μg/mL stock solution of hAChE in 0.1% (w/v) BSAand 50 mM Phosphate Buffer (pH 7.3). The organophosphorus (OP) inhibitorwas prepared in acetonitrile.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl denotes both straight and branchedgroups; but reference to an individual radical such as propyl embracesonly the straight chain radical, a branched chain isomer such asisopropyl being specifically referred to. Aryl denotes a phenyl radicalor an ortho-fused bicyclic carbocyclic radical having about nine to tenring atoms in which at least one ring is aromatic. Heteroarylencompasses a radical of a monocyclic aromatic ring containing five orsix ring atoms consisting of carbon and one to four heteroatoms eachselected from the group consisting of non-peroxide oxygen, sulfur, andN(R_(x)) wherein R_(x) is absent or is H, O, (C₁-C₄)alkyl, phenyl orbenzyl, as well as a radical of an ortho-fused bicyclic heterocycle ofabout eight to ten ring atoms comprising one to four heteroatoms eachselected from the group consisting of non-peroxide oxygen, sulfur, andN(R_(x)).

The term AChE-OP includes adducts formed when an organophosphorous (OP)agent binds to a residue (e.g. a serine residue) in the catalytic triadof the active site of AChE.

The term aged-AChE adduct includes adducts formed when an AChE-OP adductexperiences a solvolytic loss of an alkyl group.

The term reactivation (or resurrection) describes the process whereby anAged-Adduct is converted to a corresponding AChE-OP Adduct (e.g. using acompound or method of the invention).

When X is a counter ion it is understood X can be any suitablecounterion. Even if it lacks significant AChE resurrecting activity, asalt of formula I can be useful as an intermediate for the preparationor purification of other salts of formula I that have AChE resurrectingactivity. Accordingly in the compounds of formula (I), X can be anycounterion. For example, in an embodiment of the invention X can be⁻BF₄, CF₃SO₃ ⁻, F⁻, Cl⁻, Br⁻, F⁻, monobasic sulfate, dibasic sulfate,monobasic phosphate, dibasic phosphate, or tribasic phosphate, NO₃ ⁻,PF₆ ⁻, NO₂ ⁻, carboxylate, C_(e)F_(f)SO₃ ⁻, (where e=2-10 and f=2e+1),or arylsulfonyl, wherein aryl is optionally substituted with one or moregroups independently selected from halo, cyano, nitro, carboxy, hydroxy,(C₁-C₃)alkyl, (C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl,(C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio can optionally be substitutedwith one or more halo.

An aged-AChE adduct includes enzyme bound phosphonates as describedherein that have experienced a solvolytic loss of an alkyl group (forexample, see FIG. 1: R=isopropyl for sarin, pinacoyl for soman). Theaged adduct can be a phosphonate ester monoanion, which is intrinsicallyless reactive as an electrophile that the erstwhile neutral phosphonateester of the initial phosphonyl-AChE adduct.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

Specifically, (C₁-C₃)alkyl can be methyl, ethyl, propyl, or isopropyl;(C₁-C₃)alkoxy can be methoxy, ethoxy, propoxy, or isopropoxy;(C₁-C₃)alkanoyl can be formyl, acetyl, or propanoyl;(C₁-C₃)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, or isopropoxycarbonyl; (C₁-C₃)alkylthio can bemethylthio, ethylthio, propylthio, or isopropylthio; (C₂-C₃)alkanoyloxycan be acetoxy or propanoyloxy; aryl can be phenyl, indenyl, ornaphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl,oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl,pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl(or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (orits N-oxide).

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, e.g., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationsmay contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should typically be sterile, fluid and stable underthe conditions of manufacture and storage. The liquid carrier or vehiclecan be a solvent or liquid dispersion medium comprising, for example,water, ethanol, a polyol (for example, glycerol, propylene glycol,liquid polyethylene glycols, and the like), vegetable oils, nontoxicglyceryl esters, and suitable mixtures thereof. The proper fluidity canbe maintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, e.g., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Compounds of the invention can also be administered in combination withother therapeutic agents, for example, other agents that are useful forthe treatment of organophosphorous poisoning.

When the compounds of the invention are administered to counteractorganophosphorous poisoning, for example in a chemical warfaresituation, they may be self-administered, for example, by means of asyringe or a spring-loaded syringe. When the compounds of the inventionare administered as a combination with other agents (e.g. 2-PAM,atropine, and/or diazepam), the combination may also be administered bya single injection device such as, for example, a syringe or aspring-loaded syringe capable of simultaneously administering the one ormore of the agents.

Synthesis

The synthesis of the N-methyl-methoxypyridinium compounds can beconducted by exposure of starting pyridines to trialkoxonium (e.g.trimethoxonium) tetrafluoroborate or another alkylating agent (e.g.MeOTf) in a suitable solvent (e.g. methylene chloride or toluene) beforeor after oxime formation as illustrated in Scheme 1.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Synthesis of the Compound (100)

A 3 mL glass vial was equipped with a rubber septum and magnetic stirbar. The vial was brought into a glove box and charged withtrimethyloxonium tetrafluoroborate (198 mg, 1.33 mmol). The vial wassealed and removed from the glove box. A separate 3 mL vial was chargedwith 2-methoxy-6-pyridinecarboxaldehyde (Aldrich #662933, 184 mg, 1.34mmol) and was dissolved in CH₂Cl₂ (1.5 mL). The solution of pyridine wasadded via syringe onto the solid trimethoxonium tetrafluoroborate atroom temperature. The vial which contained the pyridine was rinsed withCH₂Cl₂ (1 mL) and the rinse solution was injected into the reactionvial. The reaction vial was kept at room temperature and stirring wasmaintained at ca. 400-600 rpm. Over the course of the reaction (17 h),the solid trimethoxonium tetrafluoroborate gradually dissolved, thesolution clarified, and an oil gradually formed. At the end of thereaction, hexanes (2 mL) were added after which stirring was stopped.Any solid or oil was allowed to settle and the solvent was removed byglass pipette. The solid or oil was then rinsed with several portions ofhexanes followed by diethyl ether. Residual solvent was then removed invacuo to provide the compoundN-methyl-6-methoxy-2-pyridinecarboxaldehyde tetrafluoroborate. This oilwas used for oxime formation without further purification.

A flask was charged with NaOCH₃ (18.5 mg, 0.34 mmol) and hydroxylaminehydrochloride (31 mg, 0.44 mmol) and the solids were taken up in MeOH (2mL) and left at room temperature for 5 min. The preceding aldehyde (80mg, 0.34 mmol) was dissolved in MeOH (3 mL) and was injected into thesolution of hydroxylamine. After 18 hr at room temperature, the reactionwas diluted with ethyl ether, filtered through sand, and concentrated invacuo. The residue was taken up in acetonitrile and filtered throughcelite and concentrated in vacuo. The residue was taken up in acetoneand slow diffusion of hexanes into the solution provided compound 100(37 mg, 43%) as a crystalline salt.

Example 2 Synthesis of the Compound (101)

A 3 mL glass vial was equipped with a rubber septum and magnetic stirbar. The vial was brought into a glove box and charged with MeOTf (116μL, 1.06 mmol) by micropipette. The vial was sealed and removed from theglove box. A separate 3 mL vial was charged with4-methoxy-2-pyridinecarboxaldehyde (Astatech C10253, 145 mg, 106 mmol)and was dissolved in PhMe (1 mL). The solution of pyridine was added viasyringe onto the MeOTf at room temperature. The vial which contained thepyridine was rinsed with PhMe (0.5 mL) and the rinse solution wasinjected into the reaction vial. The reaction vial was kept at roomtemperature and stirring was maintained at ca. 400-600 rpm. After 20 h,hexanes (2 mL) were added after which stirring was stopped. Any solid oroil was allowed to settle and the solvent was removed by glass pipette.The solid or oil was then rinsed with several portions of hexanesfollowed by diethyl ether. Residual solvent was then removed in vacuo toprovide the compound N-methyl-4-methoxy-2-pyridinecarboxaldehydetriflate (300 mg, 94%). This oil was used for oxime formation withoutfurther purification.

A flask was charged with NaOCH₃ (18 mg, 0.34 mmol) and hydroxylaminehydrochloride (27 mg, 0.39 mmol) and the solids were taken up in MeOH (1mL) and left at room temperature for 5 min. The preceding aldehyde (79mg, 0.26 mmol) was dissolved in MeOH (2 mL) and was injected into thesolution of hydroxylamine. After 18 hr at room temperature, the reactionwas diluted with ethyl ether, filtered through sand, and concentrated invacuo. The residue was purified by C-18 reverse phase chromatography(water/acetonitrile, 0-100%) and concentrated under a stream of air,which afforded compound 101 (18 mg, 22%) as a crystalline salt.

Example 3 Resurrection of Aged-AChE by Compound 100

Recombinant human acetylcholinesterase (hAChE) was irreversiblyinhibited by exposure to an organophosphonate (OP) analog of Sarin,7-(isopropyl methylphosphonyl)-4-methylumbelliferone (Timperley, C. M.,et al., J. Fluor. Chem., 2006, 127, 1554-1563). The fully inhibitedhAChE-OP adduct was separated on a Sephadex G-50 Quick Spin column toremove excess nerve agent, and it was “aged” for 48 hours at 27° C.Uninhibited hAChE was prepared following the same procedure bysubstituting acetonitrile in place of the OP inhibitor, and it served asthe control throughout the resurrection assay procedure. Hydrolysis ofacetylthiocholine (ATCh) by hAChE was measured spectrophotometricallyfollowing the Ellman et al. assay method (Ellman, G. L., et al.,Biochem. Pharmacol., 1961, 7, 88-95) at t=0 and 48 hours. OP inhibitedhAChE was assayed after treatment with 2-pralidoxime (2-PAM) todetermine the percentage of “aged” hAChE (% AChE_(aged)), or residualhAChE, activity as defined by Equation (1). This % AChE_(aged) is theratio of the initial rates for OP inhibited hAChE (AChE_(inhibited)) anduninhibited hAChE (AChE_(free)) catalyzed hydrolysis of ATCh.

$\begin{matrix}{{\% \mspace{14mu} {AChE}_{aged}\mspace{14mu} {activity}} = {\frac{{rate}\mspace{14mu} {of}\mspace{14mu} {AChE}_{inhibited}}{{rate}\mspace{14mu} {of}\mspace{14mu} {AChE}_{free}} \times 100}} & (1)\end{matrix}$

FIG. 2 depicts the procedural steps for a resurrection assay that can beused to determine the activity of a test compound (e.g. Compound 100).Resurrection assays were performed by preparing a 100 μL incubationsolution of 0.024 μg of “aged” hAChE in 0.1% (w/v) BSA, 50 mM phosphatebuffer (pH 7.3), and 885 μM Compound 100. “Aged” hAChE was incubated for1 hour, 4 hour, and 24 hour periods at 27° C. Following each incubationperiod, a 10 μL aliquot of the “aged” hAChE incubation solution wasassayed in 50 mM phosphate buffer (pH 7.3), 0.3 mM ATCh and 0.45 mM DTNBfollowing a 30 minute incubation with 100 μM 2-PAM and a total volume of300 μL. The percent of “aged” hAChE reactivated (AChE_(react)) by 2-PAMwas calculated using Equation (2), which is the ratio of the initialrates of “aged” hAChE (AChE_(aged)) and AChE_(free) catalyzed hydrolysisof ATCh assayed following each incubation with Compound 100. FromEquation (3), the percent of resurrected hAChE was determined.

$\begin{matrix}{{\% \mspace{14mu} {AChE}_{react}\mspace{14mu} {activity}} = {\frac{{rate}\mspace{14mu} {of}\mspace{14mu} {AChE}_{aged}}{{rate}\mspace{14mu} {of}\mspace{14mu} {AChE}_{free}} \times 100}} & (2) \\{{\% \mspace{14mu} {AChE}_{{Res}.}\mspace{14mu} {activity}} = {{\% \mspace{14mu} {AChE}_{{React}.}\mspace{14mu} {activity}} - {\% \mspace{14mu} {AChE}_{aged}\mspace{14mu} {activity}}}} & (3)\end{matrix}$

Resurrection assay data is shown in the following table for 1.77 mMCompound 100, where v_(i) is the initial rates for AChE_(aged) andAChE_(free) catalyzed hydrolysis of ATCh. Background rates were measuredin the absence of hAChE. All experiments were done in duplicate.

v_(i)(AChE_(aged)), v_(i)(AChE_(free)), Background Rate, Time, hoursmA/min mA/min mA/min % AChE_(react) % AChE_(aged) % AChE_(Res.) 1.0011.05 (±1.3) 197.50 (±8.9) 6.37 (±0.2) 2.45 (±0.7) 1.16 (±0.06) 1.28(±0.8) 4.00 16.66 (±1.0) 184.70 (±0.7) 6.56 (±0.1) 5.67 (±0.6) 1.16(±0.06) 4.50 (±0.6) 12.00 20.56 (±0.1) 191.60 (±28) 8.75 (±1) 6.46(±1.1) 1.16 (±0.06) 5.30 (±1.1) 24.00 21.90 (±1.5) 160.70 (±14) 8.68(±0.1) 8.70 (±1.3) 0.00 (±0.06)  8.7 (±1.3) 48.00 21.75 (±1.3) 213.60(±13) 7.25 (±0.3) 7.03 (±0.8) 0.00 (±0.06) 7.03 (±0.8)

All publications (including Topczewski et al., Organic Letters, 15,1084-1087 (2013), patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A method for reactivating an aged-AChE adduct comprising contactingthe aged-AChE adduct with an alkylating agent that reactivates theaged-AChE adduct to provide an AChE-OP adduct.
 2. A method for treatingan animal having organophosphorus poisoning comprising administering tothe animal an amount of an alkylating agent effective to reactivateaged-AChE to provide an AChE-OP adduct.
 3. A method for increasingacetylcholinesterase activity in an animal comprising administering tothe animal an amount of an alkylating agent effective to reactivateaged-AChE to provide an AChE-OP adduct.
 4. The method of claim 2 furthercomprising administering to the animal an agent capable of reactivatingthe AChE-OP adduct.
 5. The method of claim 4 wherein the agent capableof reactivating the AChE-OP adduct is a nucleophilic oxime.
 6. Themethod of claim 4 wherein the agent capable of reactivating the AChE-OPadduct is 2-PAM.
 7. The method of claim 2 further comprisingadministering an acetylcholine receptor antagonist to the animal.
 8. Themethod of claim 7 wherein the acetylcholine receptor antagonist isatropine.
 9. The method of claim 2 further comprising administering anantiseizure agent to the animal.
 10. The method of claim 9 wherein theantiseizure agent is diazepam.
 11. The method of claim 2 wherein thealkylating agent that reactivates the aged-AChE adduct is a compound offormula (I):

wherein: R₁ is (C₁-C₃)alkyl, aryl(C₁-C₃)alkyl, or —Y—Z, wherein any(C₁-C₃)alkyl is optionally substituted with one or more halo, andwherein any aryl is optionally substituted with one or more groupsindependently selected from halo, cyano, nitro, carboxy, hydroxy,(C₁-C₃)alkyl, (C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl,(C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio can optionally be substitutedwith one or more halo; each R₂ is independently H, halo, cyano, carboxy,—CH═N—OH, (C₁-C₃)alkyl, aryl, heteroaryl, aryl(C₁-C₃)alkyl, orheteroaryl(C₁-C₃)alkyl, wherein any (C₁-C₃)alkyl is optionallysubstituted with one or more halo, and wherein any aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom halo, cyano, nitro, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo;each X is independently a suitable a counter ion; Y is —(CH₂)_(n)— or—(CH₂)_(n)—O—(CH₂)_(n)—; n is 1, 2, 3, 4, 5, or 6; and Z is:


12. The method of claim 11 wherein X is ⁻BF₄, CF₃SO₃ ⁻, F⁻, Cl⁻, Br⁻,I⁻, monobasic sulfate, dibasic sulfate, monobasic phosphate, dibasicphosphate, or tribasic phosphate, NO₃ ⁻, PF₆ ⁻, NO₂ ⁻, C_(e)F_(f)SO₃ ⁻(where e=2-10 and f=2e+1), or arylsulfonyl, wherein aryl is optionallysubstituted with one or more groups independently selected from halo,cyano, nitro, carboxy, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo.13. A pharmaceutical composition comprising an alkylating agent capableof reactivating an aged-AChE adduct, and a pharmaceutically acceptablecarrier.
 14. The composition of claim 13 wherein the alkylating agentcapable of reactivating an aged-AChE adduct is a compound of formula(I):

wherein: R₁ is (C₁-C₃)alkyl, aryl(C₁-C₃)alkyl, or —Y—Z, wherein any(C₁-C₃)alkyl is optionally substituted with one or more halo, andwherein any aryl is optionally substituted with one or more groupsindependently selected from halo, cyano, nitro, carboxy, hydroxy,(C₁-C₃)alkyl, (C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl,(C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio can optionally be substitutedwith one or more halo; each R₂ is independently H, halo, cyano, carboxy,—CH═N—OH, (C₁-C₃)alkyl, aryl, heteroaryl, aryl(C₁-C₃)alkyl, orheteroaryl(C₁-C₃)alkyl, wherein any (C₁-C₃)alkyl is optionallysubstituted with one or more halo, and wherein any aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom halo, cyano, nitro, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo;each X is independently a suitable a counter ion; Y is —(CH₂)_(n)— or—(CH₂)_(n)—O—(CH₂)_(n)—; n is 1, 2, 3, 4, 5, or 6; and Z is:


15. The composition of claim 14 wherein X is ⁻BF₄, CF₃SO₃ ⁻, F⁻, Cl⁻,I⁻, monobasic sulfate, dibasic sulfate, monobasic phosphate, dibasicphosphate, or tribasic phosphate, NO₃ ⁻, PF₆ ⁻, NO₂ ⁻, C_(e)F_(f)SO₃ ⁻(where e=2-10 and f=2e+1), or arylsulfonyl, wherein aryl is optionallysubstituted with one or more groups independently selected from halo,cyano, nitro, carboxy, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo.16. The composition of claim 13 further comprising an agent capable ofreactivating an AChE-OP adduct, an acetylcholine receptor antagonist, oran antiseizure agent.
 17. The composition of claim 14 that is formulatedfor administration by injection.
 18. A compound of formula (I):

wherein: R₁ is (C₁-C₃)alkyl, aryl(C₁-C₃)alkyl, or —Y—Z, wherein any(C₁-C₃)alkyl is optionally substituted with one or more halo, andwherein any aryl is optionally substituted with one or more groupsindependently selected from halo, cyano, nitro, carboxy, hydroxy,(C₁-C₃)alkyl, (C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl,(C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio can optionally be substitutedwith one or more halo; each R₂ is independently H, halo, cyano, carboxy,—CH═N—OH, (C₁-C₃)alkyl, aryl, heteroaryl, aryl(C₁-C₃)alkyl, orheteroaryl(C₁-C₃)alkyl, wherein any (C₁-C₃)alkyl is optionallysubstituted with one or more halo, and wherein any aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom halo, cyano, nitro, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo;each X is independently a suitable a counter ion; Y is —(CH₂)_(n)— or—(CH₂)_(n)—O—(CH₂)_(n)—; n is 1, 2, 3, 4, 5, or 6; and Z is:


19. The compound of claim 18 wherein X is ⁻BF₄, CF₃SO₃ ⁻, F⁻, Cl⁻, Br⁻,I⁻, monobasic sulfate, dibasic sulfate, monobasic phosphate, dibasicphosphate, or tribasic phosphate, NO₃ ⁻, PF₆ ⁻, NO₂ ⁻, C_(e)F_(f)SO₃ ⁻(where e=2-10 and f=2e+1), or arylsulfonyl, wherein aryl is optionallysubstituted with one or more groups independently selected from halo,cyano, nitro, carboxy, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo.20. A kit comprising 1) an alkylating agent effective to reactivateaged-AChE; and optionally further comprising 2) an agent capable ofreactivating an AChE-OP adduct, 3) an acetylcholine receptor antagonist,and/or 4) an antiseizure agent; and 5) packaging material.
 21. The kitof claim 20 wherein the alkylating agent effective to reactivateaged-AChE is a compound of formula (I):

wherein: R₁ is (C₁-C₃)alkyl, aryl(C₁-C₃)alkyl, or —Y—Z, wherein any(C₁-C₃)alkyl is optionally substituted with one or more halo, andwherein any aryl is optionally substituted with one or more groupsindependently selected from halo, cyano, nitro, carboxy, hydroxy,(C₁-C₃)alkyl, (C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl,(C₁-C₃)alkoxy, (C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₂-C₃)alkanoyloxy, and (C₁-C₃)alkylthio can optionally be substitutedwith one or more halo; each R₂ is independently H, halo, cyano, carboxy,—CH═N—OH, (C₁-C₃)alkyl, aryl, heteroaryl, aryl(C₁-C₃)alkyl, orheteroaryl(C₁-C₃)alkyl, wherein any (C₁-C₃)alkyl is optionallysubstituted with one or more halo, and wherein any aryl or heteroaryl isoptionally substituted with one or more groups independently selectedfrom halo, cyano, nitro, hydroxy, (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio, wherein any (C₁-C₃)alkyl, (C₁-C₃)alkoxy,(C₁-C₃)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₂-C₃)alkanoyloxy, and(C₁-C₃)alkylthio can optionally be substituted with one or more halo;each X is independently a suitable a counter ion; Y is —(CH₂)_(n)— or—(CH₂)_(n)—O—(CH₂)_(n)—; n is 1, 2, 3, 4, 5, or 6; and Z is:


22. The kit of claim 21 wherein one or more of the 1) alkylating agenteffective to reactivate aged-AChE; 2) agent capable of reactivating anAChE-OP adduct, 3) acetylcholine receptor antagonist, and 4) antiseizureagent are packaged in a syringe.
 23. The kit of claim 21 wherein thepackaging material includes instructions for the administration ofthe 1) alkylating agent effective to reactivate aged-AChE; 2) agentcapable of reactivating an AChE-OP adduct, 3) acetylcholine receptorantagonist, and/or 4) antiseizure agent to combat organophosphorouspoisoning.